Publicação
Molecular mechanisms of Agrobacterium recognition and defense activation in recalcitrant plants
| Resumo: | Hypericum perforatum L. is a reservoir of high-value secondary metabolites, with a rapidly increasing interest for both researchers and the pharmaceutical industry. Hence, improving the production of these compounds via genetic manipulation is a challenging task. However, a major drawback is the recalcitrance of H. perforatum L. to Agrobacterium tumefaciensmediated transformation. Hence, in the presented study, H. perforatum L. was chosen as model plant to investigate the molecular mechanisms underlying plant recalcitrance to bacterial infection, by identifying and characterizing key genes involved in these responses. Phenolic oxidative coupling protein (Hyp-1), a pathogenesis-related (PR) class 10 family gene, was selected from a forward subtractive cDNA library from H. perforatum L. suspension cells elicitated with A. tumefaciens. The role of Hyp-1 in defense against A. tumefaciens was analyzed in transgenic Nicotiana tabacum and Lactuca sativa plants overexpressing Hyp-1, and in Catharanthus roseus silenced for the homologous Hyp-1 gene, CrIPR. Results showed that transformation efficiency of A. tumefaciens was greatly decreased in tobacco and lettuce plants overexpressing Hyp-1. However, silencing of CrIPR in C. roseus led to induction of CrPR-5 and an increased resistance to Agrobacterium infection. Moreover, the expression of core genes regulating several defense pathways was analyzed in Hyp-1 transgenic tobacco plants. Overall, overexpressing of Hyp-1 led to an ample down-regulation of key genes involved in auxin signaling (NtaTIR1, NtaAFR8), microRNA(miR)-based gene silencing (NtaAGO1, NtamiR160, NtamiR164ab), detoxification of reactive oxygen species (NtaFe-SOD), phenylpropanoid pathway (NtaPAL1, NtaPAL4 and NtaCHS) and PRs (NtaPR10 and NtaPR1). Moreover, Hyp- 1 was detected in the nucleus, plasma membrane and the cytoplasm of epidermal cells by confocal microscopy. In parallel, a Polygalacturonase inhibiting protein (PGIP) from H. perforatum L. was selected from the same cDNA library. The role of HpPGIP in defense against A. tumefaciens was analyzed in transgenic N. tabacum plants overexpression HpPGIP, and in Nicotiana benthamiana heterogeneous silenced for the homologous BoPGIP. Results showed that the transformation efficiency of A. tumefaciens was greatly decreased in tobacco overexpressing HpPGIP. However, silencing of BoPGIP in N. benthamiana led to induction of NbPRs and increased resistance to A. tumefaciens. Moreover, the expression of core genes regulating several defense pathways was analyzed in HpPGIP transgenic tobacco plants. Overall, plants overexpressing HpPGIP showed increased expression of the key genes involved in the synthesis of secondary metabolites lignin and flavonoids (NtaPAL1,4, NtaCCR and NtaCHS), ROS pathway (NtaAPX and NtaFe-SOD), as well as microRNA-mediated gene silencing pathway (NtaAGO1 and NtamiR164c). Additionally, plants overexpression Hyp-1- PGIP, showed enriched secondary metabolism and ROS defense pathways, by especially upregulating NtaCAD and NtaCAT, respectively. The expression of genes from JA signaling pathway (NtaLOX) and miroRNA-mediated gene silencing pathway (NtaAGO1) was also upregulated in these plants. In the present study, it was also demonstrated that HpPGIP protein localizes at plasma membrane, cytoplasm and nucleus. To further understand the molecular mechanisms involved in A. tumefaciens and H. perforatum L. interaction, the expression of core genes involved in plant defense pathways including secondary metabolism, gene silencing, ROS pathways and other defense genes (Hyp-1 and HpPGIP) was investigated. Results indicated that resistance of H. perforatum L. cells to infection by A. tumefaciens involves the induction of several genes encoding key enzymes of secondary metabolism, including those mediating not only the synthesis of xanthones (HpPAL and HpBPS), but also hypericin (HpOKS and HpPKS2) and melatonin (HpIGPS), ROS pathway (HpAOX) and other defense genes (Hyp- 1 and HpPGIP). Moreover, transcript levels of RDR6 and SGS3 associated to the gene silencing pathway, sense-post-transcriptional gene silencing (S-PTGS) and trans acting siRNAs (tasiRNA) pathway, together with HpAGO5, were not affected during the defense response. This study was complemented by quantifying the expression of these defense genes in H. perforatum L. plants with or without dark glands, which are known to accumulate large amounts of secondary metabolites, hence contributing to plant defense ability. Results supported the key role of these glands in gifting the host plant with resistance to pathogens, by highly accumulating secondary metabolites like xanthone (HpPAL and HpBPS) and hypericin (HpOKS and HpPKS2), together with a stimulation of the ROS pathway (HpCAT) and of the expression of other genes directly associated to the defense response (HpHyp-1 and HpPGIP). In summary, the present study provided a better understanding on the functional roles of Hyp- 1 and HpPGIP, enlightening their role in plant defense and on the network of events underlying the molecular responses of H. perforatum L. upon interaction with A. tumefacien. |
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| Autores principais: | Hou, Weina |
| Assunto: | Ciências Naturais::Ciências Biológicas |
| Ano: | 2019 |
| País: | Portugal |
| Tipo de documento: | tese de doutoramento |
| Tipo de acesso: | acesso aberto |
| Instituição associada: | Universidade do Minho |
| Idioma: | inglês |
| Origem: | RepositóriUM - Universidade do Minho |
| Resumo: | Hypericum perforatum L. is a reservoir of high-value secondary metabolites, with a rapidly increasing interest for both researchers and the pharmaceutical industry. Hence, improving the production of these compounds via genetic manipulation is a challenging task. However, a major drawback is the recalcitrance of H. perforatum L. to Agrobacterium tumefaciensmediated transformation. Hence, in the presented study, H. perforatum L. was chosen as model plant to investigate the molecular mechanisms underlying plant recalcitrance to bacterial infection, by identifying and characterizing key genes involved in these responses. Phenolic oxidative coupling protein (Hyp-1), a pathogenesis-related (PR) class 10 family gene, was selected from a forward subtractive cDNA library from H. perforatum L. suspension cells elicitated with A. tumefaciens. The role of Hyp-1 in defense against A. tumefaciens was analyzed in transgenic Nicotiana tabacum and Lactuca sativa plants overexpressing Hyp-1, and in Catharanthus roseus silenced for the homologous Hyp-1 gene, CrIPR. Results showed that transformation efficiency of A. tumefaciens was greatly decreased in tobacco and lettuce plants overexpressing Hyp-1. However, silencing of CrIPR in C. roseus led to induction of CrPR-5 and an increased resistance to Agrobacterium infection. Moreover, the expression of core genes regulating several defense pathways was analyzed in Hyp-1 transgenic tobacco plants. Overall, overexpressing of Hyp-1 led to an ample down-regulation of key genes involved in auxin signaling (NtaTIR1, NtaAFR8), microRNA(miR)-based gene silencing (NtaAGO1, NtamiR160, NtamiR164ab), detoxification of reactive oxygen species (NtaFe-SOD), phenylpropanoid pathway (NtaPAL1, NtaPAL4 and NtaCHS) and PRs (NtaPR10 and NtaPR1). Moreover, Hyp- 1 was detected in the nucleus, plasma membrane and the cytoplasm of epidermal cells by confocal microscopy. In parallel, a Polygalacturonase inhibiting protein (PGIP) from H. perforatum L. was selected from the same cDNA library. The role of HpPGIP in defense against A. tumefaciens was analyzed in transgenic N. tabacum plants overexpression HpPGIP, and in Nicotiana benthamiana heterogeneous silenced for the homologous BoPGIP. Results showed that the transformation efficiency of A. tumefaciens was greatly decreased in tobacco overexpressing HpPGIP. However, silencing of BoPGIP in N. benthamiana led to induction of NbPRs and increased resistance to A. tumefaciens. Moreover, the expression of core genes regulating several defense pathways was analyzed in HpPGIP transgenic tobacco plants. Overall, plants overexpressing HpPGIP showed increased expression of the key genes involved in the synthesis of secondary metabolites lignin and flavonoids (NtaPAL1,4, NtaCCR and NtaCHS), ROS pathway (NtaAPX and NtaFe-SOD), as well as microRNA-mediated gene silencing pathway (NtaAGO1 and NtamiR164c). Additionally, plants overexpression Hyp-1- PGIP, showed enriched secondary metabolism and ROS defense pathways, by especially upregulating NtaCAD and NtaCAT, respectively. The expression of genes from JA signaling pathway (NtaLOX) and miroRNA-mediated gene silencing pathway (NtaAGO1) was also upregulated in these plants. In the present study, it was also demonstrated that HpPGIP protein localizes at plasma membrane, cytoplasm and nucleus. To further understand the molecular mechanisms involved in A. tumefaciens and H. perforatum L. interaction, the expression of core genes involved in plant defense pathways including secondary metabolism, gene silencing, ROS pathways and other defense genes (Hyp-1 and HpPGIP) was investigated. Results indicated that resistance of H. perforatum L. cells to infection by A. tumefaciens involves the induction of several genes encoding key enzymes of secondary metabolism, including those mediating not only the synthesis of xanthones (HpPAL and HpBPS), but also hypericin (HpOKS and HpPKS2) and melatonin (HpIGPS), ROS pathway (HpAOX) and other defense genes (Hyp- 1 and HpPGIP). Moreover, transcript levels of RDR6 and SGS3 associated to the gene silencing pathway, sense-post-transcriptional gene silencing (S-PTGS) and trans acting siRNAs (tasiRNA) pathway, together with HpAGO5, were not affected during the defense response. This study was complemented by quantifying the expression of these defense genes in H. perforatum L. plants with or without dark glands, which are known to accumulate large amounts of secondary metabolites, hence contributing to plant defense ability. Results supported the key role of these glands in gifting the host plant with resistance to pathogens, by highly accumulating secondary metabolites like xanthone (HpPAL and HpBPS) and hypericin (HpOKS and HpPKS2), together with a stimulation of the ROS pathway (HpCAT) and of the expression of other genes directly associated to the defense response (HpHyp-1 and HpPGIP). In summary, the present study provided a better understanding on the functional roles of Hyp- 1 and HpPGIP, enlightening their role in plant defense and on the network of events underlying the molecular responses of H. perforatum L. upon interaction with A. tumefacien. |
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